PCR产物纯化方法 Purification of PCR Products in Preparation for Cloning相关交流-蚂蚁淘在线

PCR产物纯化方法 Purification of PCR Products in Preparation for Cloning

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PurificationofPCRProductsinPreparationforCloning

JosephSambrookPeterMaccallumCancerInstituteandTheUniversityofMelbourne,AustraliaDavidW.RussellUniversityofTexasSouthwesternMedicalCenter,DallasExcerptedFromMolecularCloning:ALaboratoryManualThirdEdition

ABSTRACT
TheresidualenzymaticactivityofThermostableDNApolymerasesthatsurvivetherigorsofPCRcancompromisesubsequentenzymaticreactions.ThisprotocoldescribeshowtouseproteinaseKtodestroythermostableenzymesandtopurifyamplifiedDNAinpreparationforcloning.

MATERIALS
ReagentsandSolutions
Ammoniumacetate(10M) AmplifiedDNAfrompolymerasechainreactions Chloroform Ethanol Phenol:chloroform(1:1,v/v) TE(pH8.0)
EnzymesandBuffers ProteinaseK(20mg/ml) GenomicgradeproteinaseKthathasbeenshowntobefreeofDNaseandRNaseactivity.
Gels EthanolAgarosegelcontaining0.5µg/mlethidiumbromide

METHOD
PooluptoeightPCRs(400µl)containing1µgofthedesiredamplificationproduct.(SeeTroubleshooting.)IfmineraloilwasusedtopreventevaporationduringPCR,centrifugethepooledsamplesbrieflyandtransferthelower(aqueous)phasetoafreshmicrocentrifugetube. Add0.2volumeof5xproteinaseKbufferandproteinaseKtoafinalconcentrationof50µg/ml.Incubatethemixturefor60minutesat37°C. InactivatetheproteinaseKbyheatingto75°Cfor20minutes. Extractthereactionmixtureoncewithphenol:chloroformandoncewithchloroform. Add0.2volumeof10Mammoniumacetateand2.5volumesofethanol.Mixthesolutionwellandstoreitfor30minutesat4°C. RecovertheDNAbycentrifugationatmaximumspeedfor5minutesat4°Cinamicrocentrifuge.Discardthesupernatantandthenwashthepelletwith70%ethanol.Centrifugeagain,removethesupernatant,andthenallowtheDNAtodry.TheDNAmaybefurtherpurifiedbychromatographyorbygelelectrophoresis.ThisstepisrecommendedwhenprimershavebeenusedtoaddrestrictionsitestotheendsoftheamplifiedDNA.Unusedprimersandprimer-dimersshouldberemovedbeforedigestingtheDNAwiththeappropriaterestrictionenzymes(pleaseseeRemovalofOligonucleotidesandExcessdNTPsfromAmplifiedDNAbyUltrafiltration). DissolvethepelletinTE(pH8.0).AssumethattherecoveryofamplifiedDNAis50-80%anddissolvetheDNAinTE(pH8.0)atanestimatedconcentrationof25µg/ml(25ng/µl). Analyzeapprox.25ngofthepurifiedDNAbyagarose-ethidiumbromidegelelectrophoresis,usingMarkersofanappropriatesize.Checkthattheamplifiedbandfluoresceswiththeintensityexpectedofapprox.25ngofDNA.
TROUBLESHOOTING
Nonspecificamplificationproductsarepresentatsignificantlevels(e.g.,aredetectablebygelelectrophoresis).Step1Purifythedesiredproductbyelectrophoresisthroughlow-melting-temperatureagarosebeforeproceeding(pleaseseeRecoveryofDNAfromLow-melting-temperatureAgaroseGels:OrganicExtraction).

REFERENCES
CroweJ.S.,CooperH.J.,SmithM.A.,SimsM.J.,ParkerD.,GewertD.1991.Improvedcloningefficiencyofpolymerasechainreaction(PCR)productsafterproteinaseKdigestion.NucleicAcidsRes.19:184-184.[FreeFullText]WybranietzW.A.andLauerU.1998.Distinctcombinationofpurificationmethodsdramaticallyimprovescohesive-endsubcloningofPCRproducts.BioTechniques24:578-580.[Medline]

Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliABIlityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:MolecularCloning:ALaboratoryManualThirdEdition,byJosephSambrookandDavidW.Russell,©2001byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p..8.25-8.26.

Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission

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PCR产物纯化方法&nbsp;Purification&nbsp;of&nbsp;PCR&nbsp;Products&nbsp;in&nbsp;Preparation&nbsp;for&nbsp;Cloning

PCR产物纯化方法 Purification of PCR Products in Preparation for Cloning